The determination of biological potency plays a key role in the development, registration, and control of biological and biotechnology-derived products. Advances in science have affected both the way these assays are carried out experimentally and how they are analyzed statistically. During the past several years, the United States Pharmacopeial Convention (USP) has focused on public standards to modernize and expand the bioassay requirements in the 

). This work has been carried out in several expert committees of the Council of Experts, USP's standards-setting body composed of volunteer scientists from many countries of the world. 

CELL-BASED ASSAYS TO REPLACE ANIMAL TESTING

In the area of assay modernization, USP activities have focused on the replacement of animal-based assays with cell-based assays. The Biologics & Biotechnology: Proteins and Polysaccharides Expert Committee currently is evaluating several proposals for product-specific, cell-based assays with allied reference material. The intent is to place these new assays in general chapters along with the already official, animal-based tests to allow compendial users a transition without compliance challenges. By using the bioassay procedure, the cell system, and the allied reference material, manufacturers, regulatory officials, and many others will soon have a comprehensive approach to ensure up-to-date measurement of the potency of biological and biotechnology-derived products. This potency is measured in units/mass of drug substance and should be consistent for a manufacturer's product over time, across manufacturers making the same product, and when feasible, calibrated to World Health Organization international units.  

The following chapters are expected to be included in 

Journal of Standards Development and Compendial Review

 <126>. In both cases, USP will support the chapters with a reference cell line that will be available for users to license. In the case of glucagon, the primary liver cell assay is supplemented with a modern assay that is based on a recombinant cell line that expresses the human glucagon receptor in large numbers. For somatropin, USP is adding a cell-proliferation assay that uses an engineered mouse pro-B cell line that was donated to USP by a pharmaceutical manufacturer and the assay is currently being established in the USP Biologics & Biotechnology Laboratory in USP's Applied Compendial Research Department. A collaborative study is planned to establish the cell line as a reference standard.  

 general chapter to elaborate a product-specific bioassay was insulin. The 

 <121> describes the use of the rabbit glucose test both as a quantitative and as a bioidentity test. Pharmaceutical manufacturers have submitted a revision request to USP to add a cell-based insulin assay to <121>. The assay is based on embryonic fibroblast 3T3L1 cells that are triggered into differentiation to form fully mature, lipid-bearing adipocytes, and glucose uptake in the cells is measured by residual glucose determination in the surrounding medium. The Proteins and Polysaccharides Expert Committee has decided to publish the new assay first as a stimulus article in 

 35(3) [May–June], while concurrently conducting additional evaluation studies in the USP Biologics & Biotechnology laboratory and with collaborating manufacturer laboratories. Interested parties can contact the USP laboratory to participate in collaborative testing (

In addition to advances in modern bioanalytical procedures, rapid progress in computational analysis, assay design tools, and statistical understanding has required revision of 

 core compendial bioassay, the general chapter 

Design and Analysis of Biological Assays 

<111>. The comprehensive revision of the chapter was proposed recently in 

 and has initiated important public comment.

  Some key issues are: 1) the evaluation of curve similarity, 2) the use of equivalence testing as an effective statistical method in several areas of bioassay data analysis, and 3) the best means for combining data from multiple assays. In its next iteration, the text will be re-proposed as 

 <1034> and will become part of a suite of bioassay guidance chapters. The current <111> will eventually be shortened and reserved for topics appropriate for a chapter below <1000>. 

 <1033> will be proposed in the March–April 2009 issue of 

<1032> currently is in development. The entire suite of four chapters (<111>, <1032>, <1033>, and <1034>) will be accompanied by a roadmap chapter that will include a unifying glossary of terms. It is USP's goal that the chapters be accompanied by example data sets that would be made available on the web. USP ultimately may make the General Chapter suite available for download from the USP web site. 

USP held an open public discussion of the new bioassay approaches in a Bioassay Workshop in August 2008 at USP Headquarters in Rockville, MD. USP will hold a similar workshop from August 12–13, 2009, at the same venue. The 2009 meeting will seek to further engage stakeholders by expanding and building on the progress made on USP's developing collection of bioassay guidance chapters. Attendees will have an opportunity to discuss chapters <1033> and <1034> with the expert drafting group and will hear an update about the development of chapter <1032>. In addition, the workshop will again provide a forum for scientific presentations and dialog among bioassay experts in the bioassay community. Special product-specific focus areas this year include bioassays for monoclonal antibodies and vaccines. To prepare a scientifically challenging and productive event, USP would like to hear from members of the bioassay community. What topics should appear on the agenda for this August? Stakeholders can submit ideas by email to 

. Please use the subject line: "Ideas: Bioassay Workshop 2009." Information regarding registration and meeting logistics will be available from 

As ongoing advances in biochemical and biophysical characterization continue to illuminate the molecular features of biological molecules, the measurement of biological potency will help establish structure–function correlations, assist in determining of immunologic response, and elucidate the molecule's biological identity. Most importantly, stability in potency results assures practitioners and patients that they are receiving a product with consistency in anticipated therapeutic outcomes batch-after-batch over many years following market access. For multiple products under the same name, the bioassay should also assist in ensuring comparable potency across manufacturers making the same ingredient or product. To this end, USP continues to work with stakeholders to provide state-of the art bioanalytical tools for compendial scientists.   

, is vice president of Biologics & Biotechnology,   

 is chair, Bioassay Ad Hoc Advisory Panel and founding Member of Biometry Associates LLC, Union City, CA, 

, is director, Biologics & Biotechnology Laboratory,  and 

, is senior scientific fellow, all at United States Pharmacopeia, Rockville, MD, 301.816.8377, 

1. Hauck WW, et al. Summary of planned revisions to design and analysis of biological assays <111>. Pharm Forum.  2007:33(3);580–81. 

2. <111> Design and Analysis of Biological Assays. Pharm Forum. 2008 34(3):685–735. 

Articles

Columns and Departments

Modern bioassays enable more accurate potency testing.

Biological Potency Assays are Key to Assessing Product Consistency

The decline of financial support for the biopharmaceutical industry has been a huge blow to the contract research and manufacturing industries. According to the National Venture Capital Association, venture capital (VC) funding for the biopharmaceutical industry in the US dropped 46% in the first quarter of 2009, to just $577 million; it had averaged over $1 billion per quarter throughout this decade. 

That funding shortfall is being felt directly by contract research organizations (CROs) and contract manufacturing organizations (CMOs), especially those that offer early development support and process development and manufacture for clinical trial materials. We are starting to see CROs and CMOs laying off staff, and some actually closing their doors, as their business volume takes a precipitous fall. 

As bad as it is in the US, however, it is worse in the UK and continental Europe. VC funding for biotech, which has always been weaker in Europe than in North America, was down 20% in Europe in 2008. According to a recent survey by the BioIndustry Association, the biotechnology industry's trade association in the UK, 76% of companies looking to raise equity in the past 12 months had difficulty doing so, with nearly 40% of those unable to raise funding at all. The situation in the UK appears to be especially bleak because the track record there is so poor. According to one source we talked to recently, only two biopharmaceuticals developed in the UK have ever achieved commercial approval, and only one of those, UCB's Cimzia, has been commercially successful. 

Given the difficult market conditions at home, it isn't surprising that UK-based CROs and CMOs are focusing their business development efforts outside the country, especially in North America. I recently toured the operations of several CROs and CMOs in the UK and talked with their executives about market conditions and their response to them. 

Eden Biodesign, located in Liverpool, reflects the recent history of the UK biopharmaceutical industry. Originally established as a consultancy by several veterans of the biotechnology industry, Eden won the contract to operate the UK's National Biotechnology Center (NBC) in 2003. The NBC was funded by a £30-million grant from national and local government agencies to offer biotech startups and university-based researchers in the UK a facility to provide process development and early-stage research and clinical materials. Eden's founders raised an additional £5 million in working capital to operate the site.  

The NBC is a 4,100 sq. m. facility with capabilities in process development and clinical manufacturing using mammalian cell culture (up to 200 L), microbial fermentation (up to 100 L), and viral production technologies. After the facility opened in 2006, Eden worked with a broad array of early-stage companies and university-based programs, many benefiting from a £3 million government fund established to provide grant support to early-stage biopharmaceutical companies.  

The grant support program came to an end at the end of 2008, and although the UK government is establishing new programs to support biotechnology as part of its economic stimulus program, Eden is now looking beyond the UK, targeting early-stage and major biopharmaceutical companies in Europe and the North America. They have established a business development team in the US and are hosting visits from global biopharmaceutical companies. In this regard, they are following a trail blazed by other UK biomanufacturers, including the Oxford-based Cobra Biomanufacturing and Billingham-based Avecia, which get the majority of their revenues from companies located outside the UK. 

An important avenue of growth for the UK-based companies is their ability to provide access to the European market and clinical sites. The EU requires that pharmaceuticals imported into its member countries be certified as GMP-compliant by a Qualified Person (QP), a formal title for professionals who have been trained and credentialed to audit manufacturers. A number of UK-based CROs and CMOs, as well as the UK-based operations of some global companies like Fisher Clinical, Almac, and Aptuit, have taken advantage of the QP requirement to grow their UK-based operations. 

Penn Pharmaceutical Services, located in Tredegar, Wales, has built a significant service offering around the QP requirement. The company has seven QPs onsite, and provides QP release for a broad array of products and clinical trial materials imported into the UK for distribution there and elsewhere in the EU. Penn's diversified capabilities include formulation and analytical development, dose manufacturing, packaging, and consultancy. It also has a thriving business manufacturing "specials," unapproved products that are a unique opportunity in the UK. 

With limited prospects locally, Penn has established a significant business development presence in the US in recent years. It also has established an alliance with US-based clinical packager Xerimis (Moorestown, NJ) for which it provides UK-based services. 

Another strategy pursued by UK-based CROs and CMOs to broaden their market opportunities is to exploit specialized scientific capabilities. This is the path being pursued by Pharmaterials, based in Reading. Founded by a university-based scientist, Pharmaterials has specialized expertise in solid state chemistry for the analysis of the alternative polymorphs and salts of small-molecule compounds. This has become an increasingly important discipline in recent years for solving solubility challenges and for intellectual property protection. It also is working with companies to develop formulations of poorly soluble compounds. Acquired by US-based Pharmaceutics International in 2008, the company recently opened a new facility for laboratories and manufacture of early-stage clinical supplies. Business from companies outside of the UK already accounts for 60% of its revenues. 

The experience of these UK-based CROs and CMOs is instructive. They have been forced by shrinking opportunities at home to look beyond their natural markets for survival and growth. They have made the investment in time and money to build understanding of new markets and create a presence in them. As the funding crisis shrinks the base of biopharmaceutical companies, all CROs and CMOs will be forced to look beyond their traditional capabilities and customer bases if they are to survive the downturn. 

 is president of PharmSource Information Services, Inc. , Springfield, VA, 703.383.4903, 

UK-based CMOs seek opportunities in new markets.

UK Service Providers Look to the New World

The US Food and Drug Administration is stepping up oversight and enforcement activity for medical products, advising industry to tighten up on data integrity and quality control to ensure compliance. "Enforcement will increase," warned Deborah Autor, director of the Office of Compliance in FDA's Center for Drug Evaluation and Research (CDER), at the April annual meeting of the Food and Drug Law Institute (FDLI). She pointed to a rise in injunctions, investigator disqualifications, and warning letters, and advised companies to avoid the Big Stick by "being pro-active about compliance." Autor's staff has grown to 250 from 150 five years ago, which increases its ability to take regulatory action; CDER had issued almost 40 warning letters as of April 14, 2009, nearly half of the total 87 sent out in 2008. 

Principal Deputy Commissioner Joshua Sharfstein, who was acting commissioner pending confirmation of Margaret Hamburg, took high-visibility enforcement actions to illustrate that a new sheriff is in town. Soon after coming to the FDA at the end of March, Sharfstein initiated a fast and more aggressive recall of pistachio products when evidence appeared of salmonella contamination. He also responded to criticism of the FDA's process for regulating medical devices by supporting a review of research requirements for device classes.  

The global swine flu outbreak provided another opportunity for Sharfstein to display a highly proactive approach. He authorized emergency access to antiviral drugs and diagnostics, and established teams under Jesse Goodman, newly named deputy commissioner for scientific and medical programs and acting chief medical officer, to facilitate the development of new vaccines, antivirals, diagnostics, and other products needed in a pandemic.  

As former director of the FDA's Center for Biologics Evaluation and Research (CBER), Goodman has been active in vaccine development and global health issues. In addition to heading up the response to the flu outbreak, Goodman will oversee the FDA's National Center for Testing and Research and agency public health and science programs, including pediatric therapeutics, combination products, women's health, Critical Path programs, good clinical practices, and the Sentinel Safety initiative. The assignment puts several hot topics on Goodman's plate, including comparative effectiveness research, biomarker identification, clinical trial design, personalized medicine, and manufacturing science. 

Congressional leaders also seek to bolster FDA leadership and authority. The Senate Health, Education, Labor and Pensions (HELP) Committee and the full Senate moved quickly last month to confirm Harmburg as FDA commissioner, stressing the need for a strong agency to deal with the swine flu outbreak, and food and drug safety issues.  

HELP Committee Chairman Edward Kennedy (D-MA) and Sen. Charles Grassley (R-IA) also reintroduced legislation authorizing the FDA to collect user fees to support more inspections of drug and medical product makers. The measure provides authority for the agency to issue subpoenas and to detain suspect products. Domestic and foreign manufacturers of regulated products would have to register with the FDA, including producers of active pharmaceutical ingredients.  

The measure is similar to the drug section of legislation backed by Rep. John Dingell (D-MI), which is pending before the House Energy and Commerce (E&C) Committee. E&C Chairman Henry Waxman (D-CA) has indicated greater interest in enacting only the food safety portion of the bill, but even that would grant FDA subpoena authority for all regulated products.  

These legislative proposals would enhance the enforcement powers already expanded by the FDA Amendments Act (FDAAA) of 2007, which authorized fines and criminal penalties for violative activity. So far, the FDA has not brought a case under these new authorities, Autor said, "but when we need to use it, we will." She pointed to the agency's speedy action to halt production of drugs by KV Pharmaceuticals, which resulted in a recall of 175 products and blocked shipment of thousands of lots. Data integrity is critical, Autor emphasized, citing FDA use of its Application Integrity Policy against Indian manufacturer Ranbaxy.  

Autor also promised to continue the FDA's campaign to remove unapproved drugs from the market, which has affected over 200 manufacturers and 500 products, and involved action against 12 classes of drugs. In March, the FDA cited nine companies for marketing 14 unapproved narcotic products. "We still see industry as somewhat complacent" about this, she said, and wants manufacturers to understand that all drugs must have FDA approval.  

Drug safety legislative proposals and FDA initiatives reflect the impact of globalization in biotech and drug manufacturing on drug safety and quality. Such "supply chain fragmentation and geographic dispersion of drug manufacturing add new risks," said CDER director Janet Woodcock at the FDLI meeting. Foreign drug manufacturing sites now exceed registered domestic facilities, and the volume of drug products made overseas continues to rise.  

An eruption in apparently intentional contamination of imported food and drug products, including heparin, pet food, and infant formula, has prompted a more deliberate FDA effort to examine the factors behind incidents involving dilution or substitution of ingredients in drugs and foods primarily to increase profits. At a public meeting last month, agency officials heard from manufacturers and other parties on ways to predict, prevent, and address economically motivated adulteration (EMA). Attendees discussed which types of products are more vulnerable, how manufacturers track and verify product components and ingredients, where supply chains are open to abuse, and what analytical methods can best detect contamination.  

Randall Lutter, FDA deputy commissioner for policy, defined EMA as the "fraudulent, intentional substitution or addition of a substance in a product for the purpose of increasing the apparent value of the product or reducing the cost of its production, i.e., for economic gain." This may involve diluting or watering down products, as in increasing the inactive ingredient in a drug. The FDA noted that dramatic shifts in product supply, perhaps to additional companies or new regions, could signal fraudulent activity. EMA may involve expensive biologics that are relatively easy to engineer, or less costly drugs that are widely used. At the public meeting, academic experts described models able to detect shifts in supply chains that can signal fraudulent activity. Roger Williams, CEO of the US Pharmacopeia (USP), urged broader FDA and industry support of efforts to update USP monographs that set a standard for product quality. The USP was in the process of revising the heparin monograph when the contamination crisis hit, Williams said, noting that "that revision came too late."  

Manufacturers backed wider use of accredited third-party inspectors to audit foreign producers of active and inactive ingredients, required registration of all ingredient producers, and stiffer penalties for drug counterfeiting and adulteration. Martin Van Trieste, vice president for quality at Amgen and a member of the Pharmaceutical Research and Manufacturers of America (PhRMA) Quality Technical Group, voiced support for a proposed Qualified Trusted Importer Program (QTIP), a voluntary certification program for qualified and compliant importers.  

Van Trieste also described industry efforts to form the Rx-360 consortium to develop best practices for supply chain management and new approaches for supplier auditing and adulteration detection. The group held a launch meeting earlier this month to discuss audit models and methods for monitoring suspicious events. Such efforts, he noted, would benefit from the assurance that manufacturers can share information on suppliers and other issues without running afoul of antitrust laws.  

Criminal elements have entered into the drug supply chain, and it's only a question of when and where EMA will emerge again, Van Trieste predicted. The short supply of antivirals in a pandemic influenza outbreak, he commented, provides the "perfect opportunity for someone to come in and make a quick buck." The industry and the FDA, he advised, have to think like the criminals to detect adulterated products.  

Washington editor, Chevy Chase, MD, 301.656.4634, 

Agency officials promise swift action against violators of drug safety and quality regulations.

Stiffer Enforcement at the FDA

The president's request for a 19% increase in the US Food and Drug Administration's 2010 budget continues the positive trend begun last year of gradually expanding resources for this underfunded agency. The additional $166.4 million for drug regulation will allow for 346 more full-time employees, an important improvement. In seeking sufficient resources for the FDA's ever-expanding responsibilities, however, the request includes both good proposals and bad ones. 

The positive side includes requests for several smart new user fees. Requiring user fees for generic drug filings, for example, is an excellent idea. Generics account for 70% of all US prescriptions, up from 50% four years ago, and the number of generic drug applications has tripled since 2001.  

In addition, these products, or at least their ingredients, increasingly are made abroad; the number of Indian facilities named in generic drug applications grew from eight in 1992 to 963 in 2008. Inspecting these foreign facilities is challenging and costly. As the FDA develops its fledgling partnerships with foreign regulatory agencies and expands its new offices abroad, ensuring quality at foreign plants should become more effective and cost-efficient in the long run. But in the meantime, the FDA needs resources to handle the generics workload on its own. These fees, furthermore, should not be tied to review timelines like those in PDUFA. The monies are needed to catch up, not speed up. 

On the negative side is a request for funding to enable the FDA to "develop policies to allow Americans to buy drugs approved in other countries." I believe this effort is misguided.  

I don't blame the FDA for this idea; it is only trying to prepare itself for what Congress may send its way. But rather than accepting drug importation as inevitable, the agency should continue to oppose it, as it has in the past. The importation of foreign drugs simply offers too many opportunities for malfeasance in the supply chain.  

As it is, the agency is only scratching the surface in terms of monitoring imports of drugs already approved for sale in the US. To see this, one only need examine the rest of the budget proposal. With the funds requested, the agency plans to double the number of drug import inspections from 2,870 to 6,197, and to increase the number of import samples analyzed from 346 to 586. Yet even with these increases, the agency would still inspect only 2% of all import lines. These numbers make it clear how few incoming shipments of foreign drugs the agency would effectively be able to monitor. 

In the budget, the agency acknowledges that a big part of its effort to improve supply chain safety will be to hold the industry accountable, recognizing that even a well funded agency could not police every move in the global pharmaceutical supply chain. (Fortunately, many industry members also recognize this, and a few have formed a new coalition, "Rx-360," to harness industry resources to tackle the challenge.)  

Access to medicine needs to be improved, but drug importation is not the way to do it. The risks are too high, and it burdens the over-stretched FDA unfairly. Properly funding the FDA to handle the ever-increasing load of generic drug reviews is a better way to ensure affordable medicine with better guarantees of quality.  

The FDA should continue to ask for the budget increases it needs. It should not, however, volunteer for new burdens that only stretch valuable resources even further for tasks that are next to impossible. 

Importing foreign drugs is not the best way to increase access to medicine. The risks are too high, and it burdens the over-stretched FDA unfairly.

The Ups and Downs of the FDA Budget

Maintaining product stability during the various drug product process unit operations is paramount to our ability to supply safe and efficacious biotech products to patients. New technologies are helping us ensure that we meet these challenges successfully and are able to embrace the Quality by Design paradigm. This article presents best practices to meet three of the significant technical challenges experienced in drug product manufacturing, namely, maintaining product stability during frozen storage, performing visual inspection of drug product vials, and controlling protein particulates. 

Drug product manufacturing has its share of operational and technical challenges. The large number of stock-keeping-units (SKUs) that we typically manufacture for a single product, as well as the need for the product to move outside the manufacturer's network and be delivered to the patient, add to the operational complexity of drug product manufacturing. Further, technical challenges associated with maintaining the purity, activity, and efficacy of the final product during drug product processing must be overcome successfully.

Elements of Biopharmaceutical Production 

series and presents best practices to meet three of the significant technical challenges experienced in drug product manufacturing, namely, maintaining product stability during frozen storage, performing visual inspection of drug product vials, and controlling protein particulates.   

Protein stability can be affected by a multitude of factors that interplay during the manufacturing of biotech drug products. The need to examine product stability over a broad range of process parameters has been highlighted in the literature.

 Such an examination can be achieved by characterization studies at small scale using qualified scaled-down models or large-scale experiments designed to examine worst-case scenarios related to changes in operating conditions.

  The development of a design space in the context of developing, scaling up, and transferring freeze-dried products has been discussed in recent publications.

  It has been pointed out that when doing formulation and initial cycle development, the development scientist must be aware of the type of equipment to which the product will be transferred in the next stage of the product lifecycle. 

Freezing and thawing large volumes of bulk protein solutions has become an important step in biopharmaceutical manufacturing because the flexibility it affords makes it possible to maximize productivity and align drug product logistics with market demands.

  The stability of therapeutic proteins during long-term storage has been highlighted as a key issue for product safety and efficacy.

  Storing drug substance for periods of time in the frozen state enables a decoupling of drug substance manufacturing from drug product manufacturing. A successful operation, therefore, requires an understanding of the fundamental aspects of freezing and thawing proteins as well as the impact of the practical aspects of heat and mass transfer, along with knowledge of the technology available.  

Manufacturing sterile biotech products requires visual inspection of the final drug product filled in sealed containers to ensure there is no contamination from foreign particulates.

  Such inspection can be performed by humans or through an automated inspection machine (AIM). Compared to manual inspection, automated visual inspection (AVI) offers more consistency, higher speed, and improved quality of inspection. It also is cost efficient over a longer period and for higher production volumes.  

Problems arising from insoluble aggregate formation in biologics development along with approaches to detect and characterize the aggregate species have been a focus for regulators and the biotech industry lately. It has been suggested that our understanding of aggregation pathways and how to inhibit aggregation remains relatively poor and that it is challenging to characterize the whole size range of particulates for a given biologics formulation.

 set limits and cite enumeration methods for sub-visible, foreign particulate matter in parenteral products.

  A significant presence of particles, whether they are product-related or foreign, may not only compromise the efficacy or the drug product but also present a safety issue.

  One important factor is potential immunogenicity, which can be a result of poor quality of the protein product.

  The quality of a product can be affected by the presence of various degradation products such as particulates and aggregates and also by chemical modifications of the protein molecule.  Protein particles typically are a result of the aggregation of structurally altered monomers and or dimers, resulting in the insolubility of the species. Aggregates or multimers can be categorized as either large or small in size.

  Small aggregates can range from dimers to multimers that can be detected by size exclusion chromatography and dynamic light scattering, with a size range of 0.1 to 1 μm. Larger aggregates can be classified as sub-visible which are 2 to 100 μm in size and detected by light obscuration methods such as the HIAC Royco liquid particle counter and microscopy. Visible particles are detected by the naked eye and can either be visible (>40 μm) or sub-visible and typically are detected by visual methods or light obscuration instruments, respectively. The size range of the protein particle can vary from <1 μm to >400 μm. In the sub-visible size range, injectable liquid formulations must comply with the pharmacopeial limit of: not <6,000 particles for the 10 μm range and not <600 particles for the 25 μm range.  

ENSURING PROTEIN STABILITY DURING FROZEN STORAGE

Freezing biologics at large-scale is carried out in various ways, from improvised to custom-designed systems. The simplest storage method involves filling the bulk solution into bottles or carboys of appropriate size and storing in freezers. These containers are often made of polyethylene or polypropylene, although steel (e.g., SS316L) can be used for small volumes. Their advantage is simplicity. Disadvantages include a lack of active control and potential variability between containers, as well as multiple container–closures to secure against contamination. The procedure for preparation, loading, and placement in the freezer has to be well defined to reduce this variability. Thawing is generally performed by placing containers in a refrigerator or at room temperature. In the absence of an active thawing mechanism, thaw times can be quite long (possibly days) depending on the size of the container. During this period, significant concentration and temperature gradients can exist in the container if it is not actively shaken or agitated. Practical handling considerations limit the size to about 20-L carboys, although 50-L sizes are possible. The system is simple, however, and if the protein formulation is stable under a wide range of freeze–thaw conditions and can withstand cryoconcentration, the bottle or carboy system may be the preferred mode of operation. 

Another solution that is available for freezing protein solutions at large scale uses stainless steel vessels (cryovessels) from Sartorius-Stedim Biotech (Aubagne, France). These cryovessels are available in multiple sizes (125-L, 200-L, and 300-L) and consist of a jacketed stainless steel tank with an internal radial finned-heat exchanger. This effectively divides the tank into six (or 10 for 125-L) longitudinal sections and has the effect of reducing the heat-transfer distance and improved heat transfer across the entire volume. Dendritic ice formation is promoted, thus avoiding the potentially damaging effects of cryoconcentration.

  The vessels are cooled and heated by an external refrigeration system that circulates heat transfer fluid through the jacket and fin system. The temperature profile of the heat transfer fluid is programmable and results in reproducible temperature profiles in the vessel. The vessel is kept stationary through the freezing process below 0 °C, but is gently agitated by rocking during the thawing process. The lack of agitation during freezing prevents solutes from moving and promotes the formation of dendritic ice. Agitation during thawing promotes rapid mixing of the thawed material, thereby removing concentration hot spots and maintaining uniform temperature in the solution with rapid thawing. The lowest working temperature for the equipment is –60 °C. 

A variation on the bulk freezing technology is the FreezeContainer from Zeta Holdings (Styria, Austria). Jacketed vessels (currently limited to 300-L) are cooled or heated through an internal circulation system (mounted in the lid). Heat exchange is accomplished by an external refrigeration system by a circulating heat transfer fluid. The temperature profile is programmable. The entire container is agitated during thawing. 

A large-scale bag freezing system called Celsius from Sartorius Stedim Biotech uses upright bags made of Stedim71 film (ethylene vinyl acetate product contact material) that are filled with the solution to be frozen and held with slight compression between two plates that serve as heat exchange surfaces. These plates are cooled or heated by circulating heat transfer fluid from an external programmable refrigeration unit. The slight compression provides improved contact and heat transfer resulting in a frozen bag in the shape of a pillow. The bag is kept in frames so as not to stress the material during handling and transport. The sizes of nominal bags are 16.6 L and 8.3 L, with fill volumes ranging between 4.2 L and 16 L, and 2.1 L and 8 L, respectively. Six bags can be simultaneously processed in the cryo unit. 

The freeze and thaw behavior of proteins has been studied extensively, but primarily in small or microscopic volumes and often in conjunction with lyophilization. The use of these small volumes in literature studies makes the process aspects difficult to relate to the freezing and storage of bulk proteins. A few studies have, however, elucidated fundamental aspects of the impact of freezing on protein structure and interaction with ice and are reviewed by Bhatnagar, et al.

An unavoidable feature of freezing is cryoconcentration as water converts to ice and excludes the solutes (and protein), ultimately creating a viscous glassy matrix (Figure 1). This can affect the embedded protein in a number of ways. If the buffer salts are prone to crystallization because of saturation, significant pH shifts can occur. Among the common buffers used for biologics, the sodium phosphate buffer mixture is particularly susceptible, and the pH can change from seven to near four on precipitation of the dibasic salt; the actual value is dependent on strength and rate.

  Even if the salts do not precipitate, buffer pH is sensitive to temperature, and therefore, pH shifts will occur during freezing and in the frozen state. Other excipients in the formulation can also cryoconcentrate. Although there is a complex dependence on factors such as the rate of cooling and composition, phase and state diagrams provide some insight into the cryoconcentrated system. If sodium chloride (NaCl) is present, a eutectic is formed at –21.2 °C which has a concentration of 23.3% w/w, i.e., an approximately 25-fold increase from 0.9% w/w normal saline. For most carbohydrates (including disaccharides), the concentration of solute in a maximal freeze-concentrated glass is around 80% w/w.

  Reactions that could lead to incompatibilities in the matrix are slowed down because of the low temperature, but the cryoconcentration of solutes can counteract this effect. Reactions such as oxidation can be enhanced, especially because the solubility of oxygen increases as temperature drops, while ice formation also excludes gases. Other potential incompatibilities among the solutes, including the protein, can be exacerbated. Proteins also interact with the ice surface with a consequent perturbation of their native structure. Proteins can partially denature at the ice interface through weakening of hydrophobic bonds as well as adsorption on the ice surface.

  This phenomenon is largely reversible after thawing, although some fraction of the protein may become irreversibly damaged. More importantly, depending on the storage temperature (in relation to the glass transition temperature of the cryoconcentrated mass), this loss of protein structure can result in aggregate formation because the partially unfolded molecules interact with other species around them. Storage above the glass transition temperature (Tg') of the matrix will allow greater mobility for this to occur. Similarly, other solutes (e.g., NaCl, glycine, mannitol, sorbitol) can phase separate, crystallize, or undergo phase transitions over time if frozen into nonequilibrium states during the freezing process, leading to protein destabilization.

  Maximally freeze-concentrated carbohydrate solutions relevant to biologics formulation tend to have a Tg' below –30 °C.

  Less than maximally freeze-concentrated systems have even lower Tg' levels. Proteins themselves have Tg' levels in the range of –10 to –15 °C, but freezing without cryoprotectants is generally not viable.

  Practical storage areas always have a degree of temperature variability within which they are controlled. Temperature fluctuations, especially in the vicinity of and above the Tg', can be especially detrimental because the rates of the processes described above will increase significantly more than would be expected based on the nominal storage temperature. 

The large-scale storage systems discussed here attempt to control the rate of heat removal and thereby obtain a reproducible process. In the case of bottles or carboys, the exact pretreatment and placement of the containers and load in the freezer must be defined. Similarly, the thawing conditions and placement must be established. The active systems from Sartorius-Stedim can be programmed to provide reproducible temperature profiles in the bulk. The actual profile is determined by the load, but a range of loads (fill volumes) can be defined, qualified, and validated. During operation, after a small degree of supercooling, ice formation is generally nucleated throughout the bulk, although the growth is faster at the edges than in the center. Depending on the rate and nature of ice growth in relation to diffusion rate, solutes get trapped between growing ice crystals. Thus, concentration gradients are generated and "frozen-in" in such systems. An example of concentration gradients observed in bottles is shown in Figure 2(a). Such gradients can persevere if thawing is carried out without mixing, as shown in Figure 2(b). Our in-house observations show that proteins and other excipients cryoconcentrate to the same extent. Cryoconcentration occurs in the cryovessels and bags.

  Because a degree of cryoconcentration is unavoidable and the protein is most vulnerable when the solute concentration is high but the mass has not been completely immobilized, it is best to freeze as rapidly as possible. Doing so minimizes the time the protein spends in the partially frozen high concentration but still mobile transition region. 

Once processed, these bulk containers must be stored for a period of time. The storage temperature is determined by the nature of the formulation as well as practical and logistical considerations. Bottles and carboys can be placed in deep freezers at any desired temperature that can be tolerated by the material of construction. The glass transition temperature of high density polyethylene (HDPE) is –145 °C for the amorphous portion (brittle temperature quoted as –100 to –70 °C), making it suitable for most applications.

  For polypropylene (PP), the glass transition temperature ranges from –15 to –10 °C requiring that such containers be handled carefully in cold storage. Similar care is required for containers based on ethylene vinyl acetate (EVA), which has a transition around –15 °C although the brittleness temperature for film-grade EVA is stated to be as low as –75 °C or below. Large stainless steel cryovessels have an operational temperature limit around –60 °C, although custom-built warehouses are required if storage below –20 °C is needed. 

The process of thawing, while simple in principle, must be controlled properly to ensure that the wall temperatures at the heat transfer surfaces do not exceed allowable limits for the product. To ensure that the thawed material does not overheat while a remainder is still in the frozen state, the mass should be agitated during processing, thereby ensuring efficient heat transfer as well as preventing hot spots. Finally, a comparable system to perform long-term stability studies is required to support regulatory filings. In the case of a bottle or carboy storage, smaller units are used. Simple dimensional considerations imply that these cannot be completely representative. For the cryovessels and bag freezing systems, small-scale models are available to do process development. Their utility as stability models must be evaluated. 

Various light transmission or camera-based commercial systems are currently available in the market and can be used to perform automated visual inspection (AVI) of sterile drug products.

  The automated inspection machine (AIM) used in this study uses a light transmission–based static division system to detect particles of foreign contaminants in vials filled with liquid product. The vial containing the product is spun at a specified speed followed by the application of brakes to stop the rotating vials. As the vial stops, the particles continue to stay in motion while being suspended in the liquid, thereby causing interference in the incident light that is detected by the sensor. The performance of an automated visual inspection system should be qualified and characterized before its usage for inspecting drug products filled in sealed containers such as vials and syringes. A Knapp study can be conducted to establish the human capability for visual inspection and to set the performance acceptance criteria for the AIM.

  In the case study presented here, process parameters that affect the performance of the AVI system, such as machine parameters, product formulation, and fill configuration were evaluated. A standard vial defect set was prepared by seeding filled clean vials with a single glass bead of size 70 μm, 100 μm, and 400 μm. Each seeded vial contained only one glass bead of a specific size. Two vial sets comprising 24 vials each (six clean, and six of each particle size) were run through the AVI system 32 times and the detection results (accept/reject) for each vial were recorded. At the end, percent detection rate (% DR) of the machine for each vial was evaluated as the ratio of the number of times the vial was rejected by the machine and the number of times the vial was inspected. Studies using product mimic solutions were designed to evaluate the effect of each process parameter on the defect detection rate by the machine.  

Key machine parameters affecting the process performance include spin speed (how fast the vial is spun, measured in rpm), brake setting (how quickly inspection is performed after applying the brakes), sensitivity (signal to noise ratio), inspection view height (based on liquid meniscus height), and background light intensity. Sensitivity and background light settings were optimized and held constant throughout this study. Inspection view height was altered based on meniscus height. Various formulations of different viscosities were then tested by changing the brake and spin speed settings. Figure 3 shows that increasing the spin speed improves the detection rate, the impact being more significant for high viscosity products. Detection of the glass particle by the machine requires the particle to move and stay suspended in the detection window. Higher spin speeds transfer more momentum from vial to liquid and then from liquid to the glass particle, thereby resulting in larger particle movement over longer durations. Inspecting the vials quickly after applying the brakes (a higher brake setting) also seemed to help the detection rates. 

In addition to machine parameters, the product formulation also can have a significant effect on the ability of the machine to detect particles. Similar to the observations in Figure 3, we observe deterioration in the detection rate as product viscosity increases. For low viscosity solutions, a spin speed of 1,600 rpm is sufficient to achieve detection rates of >80%. However, for high concentration products with viscosities >4 cP, process performance deteriorates significantly at low spin speeds. Higher spin speeds of 2,200 and 2,800 are needed to achieve the same detection rate. Other product properties such as density (relative to defect particle density) and surface tension also may affect the performance of an AVI system. Figure 4 compares the detection rate (average of 100 μm and 400 μm) of the machine for two mimic solutions of equal viscosity (2.3 cP) but different surface tensions. The formulation without polysorbate (PS-20) had a higher surface tension and was more challenging for the AIM than the one with polysorbate for the detection of 400-μm particles. The results highlight the importance of using a representative mimic solution that mimics not only a product's viscosity but other properties as well. 

The fill configuration of the product SKU also affects AVI performance. The automated inspection of syringes can be more challenging than vials because the smaller radius of syringe barrels reduces the momentum imparted to particles for a given spin speed. The fill volume of the liquid in the container also can have a bearing on the ability of the machine to detect particles. Figure 5 compares the machine performance for low and high fill volumes for two different vial sizes. For both 5 cc and 10 cc, the detection rate (plotted as the average of 100 μm and 400 μm) is lower for very low fill volumes. When the fill volume is reduced, the inspection window available to the sensor becomes smaller, thereby reducing the detection rates. Intermediate fill volumes did not impede performance as much as the low fill volumes. 

The automated visual inspection of biopharmaceuticals is a key process step in the fill–finish process and offers several benefits over manual inspection, including higher speed, better detection, and improved process consistency. The machine should, however, be qualified and characterized before its usage for product lot inspection. Machine parameters, product properties, and fill configuration are all important factors that determine the performance of the AVI system. In addition to actual product, appropriate mimic solutions can be used to characterize the effect of these parameters and design an AVI process that is robust and consistent.  

PARTICULATES IN BIOPHARMACEUTICAL FORMULATION

Particles may be generated as a result of large-scale manufacture or because of an inherent property of the protein molecule. The large-scale manufacturing of protein drug products involves processing steps such as purification, formulation, freeze–thaw, filling, shipping, and storage. Stresses that are introduced during these steps can cause instabilities that can lead to aggregation and particulation.

In this case study, we discuss some important factors that may be used to control particulation for a monoclonal antibody product. The data presented are from the formulation development of a monoclonal IgG2 antibody. A typical example of visual particulation in a biopharmaceutical liquid formulation is shown in Figure 6. 

The particles in these formulations can be counted by instruments such as the HIAC Royco liquid particle counter and characterized by purifying the particles and subjecting them to protein analysis. During formulation development, screening studies are performed to study the effect of factors such as pH, buffering agents, excipients (sugars, surfactants), and protein concentration to minimize the presence of particles. 

Figure 7 shows the effect of including a low amount (0.004% by weight) of Tween 80 (polysorbate 80) on the particle counts in a particular IgG liquid formulation. Samples were stored at 4 °C for three months and then analyzed for particulates. These IgG preparations were either derived from a hybridoma cell line or a Chinese hamster ovary (CHO) cell line. In the absence of Tween, the hybridoma-derived material had higher particle counts compared to the CHO-derived material. This difference may be a result of the inherent nature of the protein molecule or differences between the two processes. A size range of 2, 5, 7.5, 10, 20, and 25 μm is shown at 10 and 20 mg/mL. As evident from the graph, the 2 μm counts are orders of magnitude higher than the other size range and should be an important consideration in the particle analysis.

Cover

How to maintain product stability and prevent particulates.

Best Practices for Formulation and Manufacturing of Biotech Drug Products

Bioprocesses have long been ubiquitous in the production of modern pharmaceuticals and drugs. Contemporary bioprocesses are being increasingly used in the production of many other products, ranging from biodegradable plastics, packing materials, and other throwaways, to non-fossil fuels such as ethanol and biodiesel, and commonly needed human spare parts such as artificial skin and cartilage. Fluorescence-based sensing technologies, which can greatly decrease overall development time, labor, and costs, become an increasingly useful tool, particularly because their use permits a degree of miniaturization, scalability, and multiplexing that was previously unavailable.

Partly because of the growing number of applications, in the last few decades "faster, better, cheaper" has driven advances in bioprocess techniques in much the same way that it has driven advances in electronics, medical care, and other competitive industries. Even minor technological improvements in the early screening stages of drug discovery can improve speed to market and increase profits. Improvements in the pilot and production stages of development lead to reduced production costs that, for a long-lived drug (the ideal goal of any such development endeavor), can lead to increased profits over the longterm.  

The initial stages of a bioprocess development cycle, whether for a drug or other product, typically require an enormous number of experiments (thousands or even tens of thousands) to select an optimal cell line and media formulation. Even then, the cell line and formulation selected is merely the best of those tested. The more combinations tested, the better the chance that the combination selected will be close to optimal. 

These experiments must be done quickly to minimize development time. This requirement mandates that the experiments be done, to the extent possible, in parallel. Until recently, this initial screening was done almost exclusively in well plates or flasks, vessels that were reasonably inexpensive and thus economically feasible for a high degree of parallelism. However, these systems run mostly blind, with little or no instrumentation to track and control relevant process parameters.  

Typically, the only data available to the research scientist are the results of offline measurements, which are labor intensive to take and produce results of questionable accuracy because process parameters are subject to measurable change during the sampling process. Consequently, little data are obtained regarding optimal process parameters; process optimization is generally left to be done heuristically in bioreactors. The latter systems are adequately instrumented and capable of measuring and controlling salient process parameters. However, because of the discrete and expensive nature of experiments run in such systems, a fairly limited number of experiments may be performed in an attempt to determine what constitutes optimal culture conditions. Even small errors in what are determined to be (in contrast to what actually are) optimal conditions can result in enormous increases in production costs over time. In addition, if the range of allowable variances in such conditions is not adequately explored and documented, the possibility exists that valuable and viable batches of product may be discarded because of small excursions from so-called ideal culturing conditions, even though the excursions may actually be of little consequence. 

Process parameters are needed even during the screening stages of development. This is illustrated in Figure 1, which shows an experiment performed with 

 in a 50-mL flask. As shown, the dissolved oxygen (DO) level reaches its minimum (and hence the cell line its viable maximum) just three hours into the run. Commonly, such an experiment would be started in a flask, which would then be left to continue overnight without interruption until morning. Offline sampling at that point in time would be misleading because it would show only the end of run conditions and provide no information as to what actually transpired during the run. 

Of course, small laboratory-scale bioreactors could be used for the initial screening of cell lines and media as well as for process optimization. However, purchasing large numbers of individual bioreactors for the screening stage would be prohibitively expensive and setting up, running, breaking down, and cleaning a large number of such bioreactors would require an enormous amount of labor. If bioreactors were to be used, the degree of parallelism (and hence the number of experiments performed in a given period of time) would be quite limited or else the time required for the initial stages of the development cycle would increase dramatically. 

In recent years, several systems have been introduced that attempt to address the conflicting needs of maximizing information while minimizing expense. Some of these systems, such as automated plate readers and similar devices, provide an adequate degree of parallelism while providing at least some information about what transpires during culturing. However, process control in such systems is generally absent or inadequate. Newer, novel systems using microreactors or chips provide increased capabilities but are quite expensive compared to traditional systems and raise the question of how best to scale up to large-capacity systems for production purposes. 

Another approach to high throughput design is typified by systems, such as Infors Profors sparged column reactors, DasGIP Fedbatch-Pro stirred tank reactors, Infors Sixfors stirred tank reactors, and Sartorius Q stirred-tank reactors. These systems incorporate a number of stirred-tank bioreactors ranging from a few to a dozen or more, ganged together. The vessels are offered in a variety of small sizes (less than a few liters) that have volumes sufficiently large to allow repeated sampling without disruption of the process. These systems address the need for measurement and control of process parameters in a parallel processing environment, and simplify scale-up issues by means of the familiar nature of the vessels. Although they provide capabilities similar to those of an equivalent number of individual bioreactors, virtually all of the high throughput systems shown economize on bench space, provide a simple, common interface for laboratory utilities, and use a single integrated control system. Moreover, each of the systems uses common and universally accepted measurement technology so that the data generated by these systems can be readily used in the scale-up process. 

The measurement technology used in these systems is both an advantage and a drawback. The advantage is that the technology is decades old and already familiar to the operator. The drawback is that the systems, while facilitating parallel processing, are quite labor intensive: setup, calibration, and teardown and cleaning times are virtually the same as they would be for multiple experiments in an equivalent number of individual reactors. The reason for this is that the systems use conventional probe-type sensors: each vessel has its own set which must be sterilized before use, calibrated in place, and cleaned again after use. In addition, these probes must be inserted into the vessel to contact the liquid to work. This limits the system's smallest practical vessel size, even if miniature probes are used. Because of this, the volumes of required media (and thus supply costs) are nearly the same as they would be if banks of individual bioreactors were used in place of these high throughput systems. Because there is no appreciable savings in supply costs or labor costs, the attractiveness of these high throughput systems is limited. 

There is another approach to high throughput design using a technology that's been known for many years, but has not, until recently, seen widespread adoption. Fluorescence-based sensing technology combines novel chemistry, optics, and electronics in the design of a sensor that's virtually calibration-free, can be miniaturized to accommodate the smallest vessels or well plates, isolates the sensing electronics from the cell culture broth, and can be multiplexed easily, if needed, to measure values of process parameters in a large number of vessels without concern for contamination. 

Figure 2 shows such a sensor schematically. The sensor consists of two major components: a sensing head that contains both optics and electronics; and a peel-and-stick sensing patch or foil that is affixed to the inside of the vessel being monitored.  

One or more high intensity light emitting diodes (LEDs), after proper bandwidth filtering, are used commercially to illuminate the sensing patch, which is prepared with a dye that causes reflected light (measured at a different wavelength) to vary in a well-defined and consistent way with changes in the measured parameter.  

As an example, a system for measuring DO can be designed based on the principal that oxygen quenches, in a dynamic and well-defined manner, the fluorescence of any fluorophore with a lifetime longer than 10 ns. Such a sensor will use a modulated, properly filtered light source (a high intensity blue LED, for example) to excite a patch treated with a specific compound, commonly a ruthenium-based or platinum-based fluorophore, although other compounds have been used.  

The patch, when excited, will reflect light, which differs from the incident light in wavelength, phase, and intensity. The amount of phase shift and difference in intensity are dependent on the DO level in the immersion liquid. The reflected light, properly filtered, can be captured using a common photodetector. Either change in intensity or shift in phase can be used as the primary measured variable, although intensity is less desirable because of possible errors caused by background noise, ambient light conditions, and changes in incident intensity caused by aging of the excitation LED and associated electronics. Although more difficult to measure electronically, phase shift is thus the preferred parameter in this case. 

, and other parameters can be designed in a similar manner. Despite the differences in optics, chemistry, and methodologies, these parameters are measured with instruments using physically similar designs and apparatus, and are based on the same principal of exciting a patch treated with a specific fluorophore with one or more light sources, and measuring and analyzing the reflected light. A pH sensor, for example, might use a patch treated with a dye such as hydroxypyrene trisulfonic acid (HTPS), which exhibits two excitation wavelengths that correspond to the acid and its conjugate base. Using a pair of high intensity LEDs to excite the patch, one ultraviolet and one blue, the ratio of emission intensities can be used to determine pH. CO2 can be measured in a similar manner. Research is currently being directed toward the robust design of sensors for more exotic parameters such as alcohols, green fluorescent protein, and glucose.  

Some care must be used in the design and fabrication of the sensing patch. Typically, such patches comprise a number of thin layers: a protective layer made of mylar or similar material to protect the adhesive until the patch is ready to be pressed in place; the adhesive layer; a support layer to provide some degree of rigidity to the patch; the sensing layer; and a cover layer that provides optical isolation for the sensing layer. The sensing layer contains the dye, which is usually immobilized in some fashion to minimize leaching of the dye into the medium. For example, for DO, the dye can be immobilized in an oxygen permeable polymer film or absorbed on silica gel. The cover layer is required to prevent ambient light from unduly affecting the sensor's readings. 

With minor modifications, fluorescence-based sensors also can be used to measure process parameters in a gas stream as well as a liquid. By using three such sensors with a bioreactor system (to measure DO in the inlet and outlet gas streams and in the culture medium), it is easy to determine total oxygen uptake rate over a period of time. If the uptake rate of the cell line is known, this provides an easy way to determine viable cell density during a culture, and thus the optimal harvesting point. 

Because there is no probe entering the culture vessel with a fluorescence-based sensor, these sensors can be used in vessels of only a few milliliters in size. This is because the illuminated area of the patch (the only component that must be placed in the vessel) need be no more than a few millimeters in diameter, or even smaller, if fiber optics are used as a light source and receiver in place of the excitation LED and photodetector. Of course, the use of fiber optics has its own unique set of problems, but it has enabled the technology to be used in applications that otherwise have very limited space for the accommodation of sensors for parametric measurement. 

Fluorescence-based technology has a number of advantages over conventional probe-type sensors. The ability to inexpensively measure process parameters in small vessels allows key operational parameters to be accurately measured much earlier in the development cycle than would be possible otherwise, such as at the flask or well-plate stage rather than the bioreactor stage. This permits early determination of optimal media formulation and cell culturing conditions, and thus facilitates scale-up from glassware to laboratory-scale and production-scale bioreactors.  

An important additional advantage of the technology (particularly in fully instrumented systems) is that sensing electronics are extremely stable, requiring only a yearly measurement of electronic offsets (a procedure that takes less than a minute), thus rendering obsolete the time-consuming calibration procedures required for the proper use of conventional probes. By virtually eliminating calibration, the amount of setup labor required to use these sensors is substantially less than with conventional probes. Moreover, because the electronics are never in contact with the process (only the sensing patches are in contact and, although they can be autoclaved, they are cheap enough to be discarded after each use), there is no need for cleaning and sterilization of sensors between experiments. Again, the savings in time (and thus the savings in labor costs) can be quite substantial. 

A final advantage of the fluorescence-based sensors is that they can be readily multiplexed so that one sensor is used to measure process parameters in many vessels. For applications in which measurements need be taken only at infrequent intervals, a sensor can be moved from vessel to vessel to measure a given parameter. In such a case, it is only necessary to provide a sensing patch in each vessel. Because the patches cost only a few dollars each, the use of a single sensing head with multiple patches greatly decreases instrumentation costs.  

Conversely, another approach to multiplexing uses robotics, X–Y tables, or rotary tables to carry vessels to fixed sensors. For example, the Fluorometrix CellStation HTBR-1 is a 12-reactor system that uses a rotary table to automatically position, at a programmed interval, each of the 12-reactors over a single fluorescence-based module that measures both pH and DO. The same scheme can be used with well plates and other similar devices. The number of vessels or wells that can be monitored with each fixed sensor depends only on the measurement time (which is generally a few seconds) and the maximum time allowed between readings. 

As demonstrated above, the use of fluorescence-based sensors during the screening phases of the bioprocess cycle can greatly reduce the elapsed time required to complete these phases and do so with great savings of labor, media, and even facilities costs. However, the greatest value of the technology is that it provides information about the process that might otherwise have to be determined during later phases of the development cycle. For this reason, its use in the screening application is in keeping with the FDA's Process Analytical Technologies initiative (PAT). The FDA encourages a rather broad application of PAT by defining it as: 

 "Tools and systems that utilize real-time measurements, or rapid measurements during processing, of evolving quality and performance attributes of in-process materials to provide information to ensure optimal processing to produce final product that consistently conforms to established quality and performance standards."

The ultimate goal of PAT is the realization of measurable process conditions that are linked directly to the quality of the product being developed. The ability to measure viable process conditions during the screening phases of the development cycle provides a wealth of information about these cell growth conditions, and therefore, allows an assessment of how closely process-screening devices can be used to replicate large-scale conditions. This data will provide a smoother transition to large-scale processes, requiring fewer experiments to be done in expensive and relatively labor-intensive bioreactors during the laboratory-scale phases. Also, it should be noted that the use of this sensing technology does not require the purchase of novel and costly systems for the screening phases. The sensors are compatible with the well plates, dishes, and flasks that have traditionally been used in small-scale screening operations, and can be readily used to retrofit existing equipment. 

Despite the focus on the screening phases of the development cycle, this sensing technology also has value during the later phases of the development and production cycle. Recent trends tend toward the use of disposables during these phases because of lower operation and validation costs.

 Disposables primarily take the form of bag type bioreactors—plastic pillows that can be charged with a few to many hundreds of liters of cell culturing media. Because they are generally shipped sterile and disposed of after use, they require less labor during setup and teardown of the culturing system than conventional stirred-tank reactors. However, these bioreactors require the same careful monitoring as the conventional bioreactors. If conventional probe-type sensors are adapted for use in these disposable bioreactors, the system is then only "semi-disposable" because the sensors must be sterilized before and after use, and calibrated during use. To address this issue, some recent efforts have been made to develop miniature disposable probe-type sensors so that the entire system becomes truly disposable. These sensors, however, are still relatively costly for a disposable system, and fail to adequately address calibration issues and the consequent labor and time requirements. 

A different approach to fully disposable systems that has been adopted by several manufacturers is the incorporation of fluorescence-based sensors in disposable-bag bioreactors.

  The sensor technology is applicable to any size bioreactor because it uses no volume-dependent components. The sensing patches themselves may be placed in the bags during fabrication and then sterilized by irradiation or by autoclaving, and then sealed into a presterilized bag. Alternately, the sensing patches can be autoclaved on a carrier (a clear glass or autoclaveable plastic slide or disk) that can then be inserted into a pocket in the bag before final sealing. This latter technique has the added advantage of facilitating alignment of the sensing patch in the bag with respect to the sensing head or fiber. The patches, which cost only a few dollars, are far less costly than even a disposable miniature probe and can be disposed of along with the bag after use. The sensing head itself remains ready to use again. In addition to the cost savings resulting from the disposal of only a very inexpensive patch, elimination of the effective need for calibration saves substantial time and labor costs associated with the culturing process. 

Given the many advantages to fluorescence-based sensing, it is only natural to ask why this technology, which is nearly three-decades old has been so slow to catch on and why, even now, its use is primarily associated with small-scale apparatus.

There are several factors that have contributed to its slow adoption. First, the biotech community is notoriously slow to embrace new technologies, particularly during the pilot and manufacturing phases of a drug-development cycle. This is because the cost of drug development and approval often greatly outweighs the cost of drug production, and the elapsed time required for drug approval is often far greater than the time required for screening and laboratory-scale operations. The use of a decades-old technology provides the manufacturer with a certain level of assurance during the approval process, and might therefore be preferred over a newer technology even if the newer technology is less costly and more efficient.  

Second, the fluorescence-based instruments are just now reaching a level of robustness and affordability that makes them suitable for use in bioprocessing. Although conventional probes are subject to inaccuracy because of drift over time, this artifact can be virtually eliminated through frequent and careful calibration of the probe, a time-consuming operation but one that is effective nonetheless. In contrast, early fluorescence-based probes had several sources of error: inherent electronic variance from sensor to sensor, variability of patch formulation from batch-to-batch, and the need to ensure that the vessel being used conformed to certain specifications. In recent years, these problems have been substantially addressed: by including a microprocessor on board each instrument, the electronics can be normalized from sensor to sensor, largely mitigating electronic variance. Patch-to-patch variation is addressed by associating a calibration code with each batch of patches, similar to the codes used with most diabetic test strips, and the measurement results have been shown to be largely independent of bioreactor-to-bioreactor variance. The referenced testing was done using the Fluorometrix CellStation HTBR-1 12 station system.

Robustness was initially not the only drawback to the technology—equally important was instrument cost. The original prototype instruments used lasers as an excitation source and were consequently financially unsuitable for practical applications. The use of high-intensity LEDs for excitation, which have in the last years dropped in price to a couple of dollars, together with efficient optical and electronic designs, addresses that problem: fluorescence-based sensors are potentially cheaper than conventional probes. Much has been done on the design of a new generation of sensing patches that can be demonstrated to not leach into the culture media or otherwise affect the growing cells.  

Even with all of these advances, the technology still has a potential disadvantage in that the sensing patches are subject to photobleaching over time. Because of improved formulations which greatly increase the lifetime of the patches, and the use of phase shift or ratiometric measurements, the sensor's measurements are immune to changes in the patch response caused by photobleaching, except after long-term use. In such a case, the signal to noise ratio drops with time until the measured signals become noisy and less accurate after an excessive number of measurements. Because the effective lifetime of the sensing patch is measured in tens of thousands of readings, this is a problem only with very long processes monitored at frequent intervals (seconds versus minutes or hours). Even in this case, an easy solution exists: a vessel can be equipped with two or more patches in place of a single patch and the sensing head can be moved to a fresh patch whenever the measurements start to become noisy. In this way, processes of arbitrary duration can be monitored with no loss of accuracy and no drift over time.  

As the need for more cost-effective and rapid development of drugs and other bioprocess-related products increases, so does the tolerance for new technologies and approaches in what has been a rather conservative industry. Fluorescence-based sensing technologies, which can greatly decrease overall development time, labor, and costs, become an increasingly useful tool, particularly because their use permits a degree of miniaturization, scalability, and multiplexing previously unavailable. Although there are limitations, particularly in patch development, continued development and maturation in this area holds great promise for the development of a widespread paradigm-shifting technology in the near future.   

 is the president and CEO of Fluorometrix Corporation, Stow, MA, 978.461.2468, 

Features

A prove-free system monitors accurately at very small scale.

Using Fluorescence-Based Sensing to Accelerate Process Development

The industry is in transition. There is a renewed focus on cost and efficiency, and as a resilt, we are seeing a major uptake in the use of disposables technologies. The opportunities provided by disposable technologies also come with risks, however, and single-use technologies are not a homogenous toolkit that provide a simple fix. Rather, the market offers a mixture of technologies at different states of maturity from a wide variety of suppliers. This variability poses challenges in terms of supply chain and technical risk. In this article, we present the end-user perspective. Since the last column, we have visited Interphex in New York, and talked to a number of industry leaders from the manufacturing and contracting manufacturing sectors for their perspectives on the real concerns arising from the adoption of disposable technologies for the commercial manufacture of biotech products.  

When we discussed the challenges and opportunities posed by disposable technologies, we asked a number of questions. Do disposables really change the way end users look at and manage operations? Do disposables technologies change the way they view and operate the factory? How can the opportunities be maximized, and the downsides be minimized?  

From these conversations, several key challenges emerged. 

There are significant differences in the announced pricing from one supplier to another. Many suppliers try to play on the fact that they have a unique technology and accordingly charge a significant premium, whereas others take a pragmatic approach, thinking about long-term relationships. One interviewee said that cost does indeed matter. "The end-user may appreciate a new disposable technology for the benefits it brings in terms of simplifying equipment preparation, cleaning, etc., but will not adopt this technology if it actually works out [to be] more expensive than the technology the end-user was working with previously," he said. 

 By moving to disposables, the end user is effectively outsourcing a major part of manufacturing and quality management. As a result, the user must work very closely with the supplier to ensure that robust quality throughout the manufacturing process is maintained. 

 Supply chain risk is a common sourse of complaints related to dual sourcing and consistency of supply. One user commented that his (her) company's zero change policy requires careful planning. "We ask our disposable suppliers contractually for a guarantee of supply without change for a 5–10 year period," he (she) said. Such a policy means that the user has to consider the supply chain in most minute detail in terms of component and film suppliers and ensure that all the necessary contracts are in place. 

One user noted the operator's role in terms of handling of large-scale containers and the manipulation of tubing sets. "When working with disposables, there is a much greater dependence on the operators themselves. If they make a manipulation error, this could contaminate the whole process," he said. "We work with our suppliers in designing disposable systems that are as intuitive as possible, are idiot-proof, and so minimize the risk of operator error." 

The opportunities presented by single-use technologies for manufacturing tend to relate to flexibility of operation, ease of product changeover, and the removal of wasted activities in terms of preparation and cleaning. "The great opportunity with disposables is that we can spend more time concentrating on manufacturing steps, the productive steps, and get rid of preparation work," said one interviewee. Users also see significant benefit from disposables when applied to process development, where fast turnaround in pilot and clinical supply facilities are extremely important. 

DISPOSABLE TECHNOLOGY EVALUATION FACTORS TO CONSIDER

The preceding section on challenges and opportunities leads us to the evaluation process. All users have to evaluate new technologies, and disposables are no different. So what are the specific considerations? As expected, this is not a simple process and many factors must be considered. It is also important to develop a close collaboration with the supplier during the evaluation process. 

The goal is to to ensure a supply of material with totally reproducible, consistent quality. How can this be achieved? End users indicated that there is still a way to go before such reproducibility is achieved. One end user indicated that during audits, his team looks carefully at the training programs put in place for the staff that manufacture the disposables systems. The level of staff turnover is also noted. "The manufacturing and assembly of disposable systems is rarely highly automated, so consistent quality can only be obtained by highly experienced manufacturing operators at the disposables manufacturing end," he (she) also explained. Of course, quality controls, over incoming raw materials and in-process, and on finished products plays an important role in ensuring reproducible quality. 

 Are there savings to be had at the time of implementation and over the whole product lifecycle? A business case and comparison with existing systems typically will be made. Many companies assess the total cost of ownership, and some use net present value models that integrate costs from each process step. According to one user, when looking at costs, "we integrate waste costs but we don't integrate the relative waste costs for traditional manufacturing technologies." In this instance, although the user is evaluating the increase in the solid waste costs of disposables, they do not evaluate the reductions seen in liquid wastes when moving from disposables.  

What is the level of maturity of the technology and the potential of risk to product? 

As discussed above, companies place enormous emphasis on the robustness of the disposables supply chain. One interviewee from a major vaccine manufacturing company reinforced this point. "We consider [that] we are taking considerable risk [when] outsourcing many activities for our manufacturing requirements that we would have previously carried out internally," he said. As a result, his team asks many questions, such as: Is this an experienced mature supplier who has been supplying disposable technologies to the market for several years, or a total newcomer? Is there an alternative supplier for a given technology? "Certainly, our aim is to have dual sourcing for all our disposable technologies," he continues. "For existing applications, this is not always the case, because validating a second supplier represents a lot of work, so the resources need to be made available to carry out the necessary validation work." If there is only one supplier of a technology, then the end user clearly needs to carry out a very detailed supply chain evaluation. 

As with any supplier, there must be regular audits and controls, and strong relationships must be built up to create trust. In terms of risk, the fact that there has been considerable consolidation among the filter and bag suppliers limits choice. Ideally, for every technology there would be the option to work with material from an alternative vendor, but that may not be possible.  

Another interviewee from a major monoclonal antibody manufacturing company explained increased supply chain risk involved in disposables and how his company handled it. "In contrast to traditional reusable systems, where we have full control in terms of ongoing maintenance and so on, with disposables we are changing the scenario and have far less control," he (she) stated. "We have developed a full deviation and investigational methodology for identified defects in the disposables supply chain as this could potentially affect a large number of our products." 

The evaluation process typically involves a feasibility study, testing, and comparison to the traditional set-up. Depending on the complexity of the technology, the evaluation can last from a few months or up to two years. Generally, the technology will initially be implemented at pilot-scale, occasionally with the need to work with the supplier to fine-tune certain technology features and to ensure the robustness of the supplier's manufacturing, quality, and supply chain for the given technology.  

There were several common issues that were experienced during this evaluation process. 

First, end users often have technical questions relating to specific features of the technology (such as the agitator not working correctly, or difficulty with the aeration systems, filters, and other systems). 

End users also often have questions about validation, and validation inevitably takes longer than anticipated. Users are often told that validating disposable systems is simpler than validating traditional systems, but this is not the experience of many of the end users we talked to. "The different studies that we need to carry out in terms of chemical resistance and interaction studies often take considerably longer than the equivalent studies required for traditional systems," commented one. 

So what are the important criteria that must be met before any disposable technology can be adopted? They are performance, cost, and supply chain security. 

Any discussion of what's missing has the implied assumption that we are trying to achieve a totally disposable process, but this is not the case. "I would not talk necessarily about gaps in the disposable technology offering—it is a more a case of where does it make sense to use disposables for a given process," said one user. "The suppliers think that they can solve everything with disposables!"  

There are a few gaps in component offerings, however, one common desire is for a mature disconnection system that can be used several times. Users recognize, however, that we are just at the beginning of the technology developments in this arena.  

Another frequent request among end-users was a smaller-scale version of the single-use stirred tank bioreactor. End users asked that such systems be made available in the 10- and 20-L format.  

It is apparent from the feedback that there is some level of dissatisfaction on the service front, with some end-users demanding:  

Disposables suppliers guaranteeing a no-change policy for a 10-year period. 

More collaboration between suppliers, leading toward greater standardization of films, containers for bags, and components used in systems. 

As the disposables market matures, there is also a requirement that the disposables suppliers think about the longer-term requirements of the industry rather than trying to impose single-source dependency. 

Indeed, the way in which the disposables suppliers approach the market suggests that different companies and technologies are at different stages of maturity. The disposables suppliers who have been supplying the industry for many years have reached a level of maturity that allows them to approach the market thinking about the requirements for industrial implementation, whereas the suppliers who have arrived on the market more recently are still approaching the market from an innovators or early adopter point of view by considering only the technical issues without considering the users' requirements from a business perspective for things such as consistent quality and supply chain security.  

If a larger number of disposables suppliers were able to reach the required level of maturity, the end-users we talked to believe that this would enable a far greater deployment of disposable systems because the risk the end users are taking would be diminished. As one user said, "Our company has quite frequently walked away from certain disposables implementations due to the lack of maturity of the supply chain and lack of availability of dual sourcing." 

It is apparent that many companies are concerned about environmental issues. This in part is being driven both by good stewardship and by legislation. Many of the companies we have talked to have internal initiatives to reduce their carbon footprint. However, there is always a question of the methodology used to determine where to start and where to finish when comparing stainless and disposables. Again, one user illustrates a fairly typical viewpoint. "We do have an internal initiative around environmental and operational sustainability," he said. "We also talk about the concept of green procurement ... the article, 'The Environmental Impact of Disposable Technologies: Can Disposables Reduce Your Facility's Environmental Footprint?' has been widely read in our organization and has generated considerable internal discussion."

What end users think about single-use systems.

BioPharm International-06-01-2009